Half‐bleach experiments were performed 24 h after transfection of the indicated constructs using the FRAP module of the Leica SP8 microscope. The Leica Application Suite X software was used for quantification and GraphPad Prism to plot and analyze replicate half‐bleach experiments. For each recorded time point (t), the fluorescence intensities within the bleached droplet hemisphere were integrated and normalized to the fluorescence intensity of the corresponding unbleached droplet hemisphere. These normalized, time‐dependent fluorescence intensities It were then used to calculate the fluorescence recovery (FR) according to the following formula: FR(t) = (It − It0)/(Ibefore bleaching − It0), with t0 being the first time point observed after photobleaching.
Frap module
Manufactured by Leica
The FRAP module is a key component of Leica's advanced imaging solutions. It is designed to facilitate Fluorescence Recovery After Photobleaching (FRAP) experiments, a powerful technique used to study molecular dynamics and interactions within living cells. The FRAP module provides the necessary hardware and software integration to enable precise photobleaching and subsequent fluorescence recovery analysis, allowing researchers to gain insights into various cellular processes.
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5 protocols using frap module
1
Fluorescence Recovery After Photobleaching
2
Microscopy Imaging of Live Cells
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For live microscopy, cells were plated on Lab-Tek-chambered 1.0 borosilicated coverglass slides coated with 5 μg ml−1 fibronectin and imaged within microscope incubators at 37 °C and 5% CO2. Widefield imaging (Figs 3d , 4b and 8a ; Supplementary Movies 2–8 ,11 and 12 ) was performed on an inverted Zeiss widefield Observer.Z1 microscope equipped with a 63 × 1.40 Plan Apochromat oil objective, definite focus system, and Hamamatsu Orca-R2 digital camera. For the Dendra2 photoswitching experiments (Fig. 6a ; Supplementary Movie 9 ), a LEICA TCS SP8 confocal microscope system equipped with a 63 × 1.4 NA oil objective, and 405 nm diode, 488 nm argon and 594 nm HeNe lasers were used. Regions of interest were photoswitched by a focused 405 nm diode laser beam illumination using the LEICA FRAP module. FRAP experiments (Figs 6b and 9f ; Supplementary Movie 10 ) were performed on a Zeiss LSM 510 Meta confocal laser scanning microscope using a 40 × 1.3 NA EC Plan Neofluar oil objective, and argon 488 and DPSS 561 nm lasers. A defined area of interest (9.64 μm2) was photobleached by 200 iterations using the DPSS laser at 561 nm at maximal power (15 mW). Images were enhanced for display with an unsharp mask filter and adjusted for brightness/contrast in ImageJ.
3
FRAP Analysis of Condensates in Arabidopsis
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FRAP experiments were performed on condensates from the pUBQ10:YFP-ARF19 (Upper root) and aff1-1 pUBQ10:YFP-ARF19 (Upper root and root tip) lines using a Leica SP8 confocal microscope. All imaging was carried out using a PMT detector and a 40 × water immersion objective under the Leica FRAP module. Imaging used a 512 × 512 format and a scan speed of 400 Hz. Method of bleaching were set as follows: fly mode – off, zoom in – on, change bleach format – off, set background to zero – on, delete bleach images after scan – off, Use laser settings for all ROIs – on. For photobleaching, the 488 nm, 514 nm and 552 nm were set to 100% power. Two pre-bleach images were acquired followed by the photobleaching and then 100 postbleach images were captured at 5.17 s intervals. After image acquisition, data was imported into FIJI (ImageJ) for further analysis.
4
FRAP Imaging of Biomolecular Condensates
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FRAP imaging was carried out on a Leica SP8 using a PMT detector and a 40 × water immersion objective using the Leica FRAP module. All FRAP imaging was carried out immediately after N&B imaging. Pre- and post-photobleaching image acquisition used a 514 nm laser at 0.06% power with a range of acquisition of 519 nm to 550 nm. All imaging used a 512 × 512 format and a scan speed of 1400 Hz. One pre-bleach image was acquired followed by the photobleaching and then 120 post-bleach images were captured at 1 s intervals. The duration per acquisition of each image was 0.371 s. For FRAP imaging, the zoom was adjusted as needed depending on the size of the condensate. All optional SP8-specific FRAP module settings were set as follows: fly mode—off, zoom in—on, change bleach format—off, set background to zero—off, delete bleach images after scan—off. For photobleaching, the 448 nm, 488 nm, 514 nm, and 552 nm lasers were set to 100% power and targeted to approximately one half of the condensate for a total of 1.8762 s. Following image acquisition, data was imported into FIJI (FIJI Is Just ImageJ) [60 (link)] in the original.lif file format for analysis. The percent recovery was determined by quantifying the amount of recovery observed in the photobleached region post-photobleaching.
5
FRAP Imaging of Biomolecular Condensates
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FRAP imaging was carried out on a Leica SP8 using a PMT detector and a 40x water immersion objective using the Leica FRAP module. All FRAP imaging was carried out immediately after N&B imaging. Pre-and post-photobleaching image acquisition used a 514 nm laser at 0.06% power with a range of acquisition of 519 nm to 550 nm. All imaging used a 512x512 format and a scan speed of 1400 hz. One pre-bleach image was acquired followed by the photobleaching and then 120 post-bleach images were captured at 1 second intervals. The duration per acquisition of each image was 0.371 seconds. For FRAP imaging, the zoom was adjusted as needed depending on the size of the condensate. All optional SP8-specific FRAP module settings were set as follows: fly mode -off, zoom in -on, change bleach format -off, set background to zero -off, delete bleach images after scan -off. For photobleaching, the 448 nm, 488 nm, 514 nm, and 552 nm lasers were set to 100% power and targeted to approximately one half of the condensate for a total of 1.8762 seconds. Following image acquisition, data was imported into FIJI (FIJI Is Just ImageJ) [60] in the original .lif file format for analysis. The percent recovery was determined by quantifying the amount of recovery observed in the photobleached region post-photobleaching.
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